Masayuki Takeuchi

Influence of Fusarium wilt toxin(s) on carnation cells

The influence of culture filtrates of Fusarium oxysporum f.sp. dianthi which causes Fusarium wilt was investigated on growth and viability of carnation tissue cultures and leaf segments. Culture filtrates of avirulent race 1 of this fungus did not affect calli and leaf segments of cultivars both susceptible and resistant to Fusarium wilt. However, culture filtrates of virulent race 2 decreased viability and suppressed growth of callus of the susceptible cultivar. In contrast, callus of the resistant cultivar showed resistance to the culture filtrates. The results of these experiments may provide information on methods of selection of new wilt resistant carnation varieties.

A study of cell wall regeneration of protoplasts inMarchantia polymorpha L.

Protoplasts ofMarchantia polymorpha L. were isolated from suspension cells. Regeneration of cell walls on the surface of the protoplasts began within a few hr of cultivation. New cell walls completely covered the surface of the protoplasts within 48 hr. Coumarin and 2,6-dichlorobenzonitrile treatment inhibited the formation of the new cell wall. In the initial stage of cell wall regeneration, endoplasmic reticula developed remarkably close to the plasma membrane in the protoplasts, but no development of Golgi bodies was observed at the same locus. This may suggest that the Golgi bodies do not play an active role in the cell wall formation, at least not in very early periods of cell wall regeneration. The development of endoplasmic reticula and an ultrastructural change of plasma membrane from smooth to rough may be important in the cell wall formation of protoplasts.

Enhancement of Cell Division in Marchanda Protoplast Culture by Activated Charcoal

Summary Protoplasts isolated from callus culture of liverwort (Marchantia polymorpha L.) were cultured in the presence of activated charcoal. The rate of cell division of regenerated protoplasts was enhanced by an addition of activated charcoal to the culture medium. Partial but considerable enhancement of cell division was observed with frequent washing of the protoplasts and replacement of the medium during the initial stage of culture. These results suggest that a certain substance(s) inhibitory to cell division and absorbed by activated charcoal is released from protoplasts and/or regenerated protoplasts.

A simple and rapid method for determining cell survival in the cryopreserved shoot apex using luciferin–luciferase ATP assay

Abstract A new method was developed to estimate the survival rate of cells in the shoot apex using an ATP assay with luciferin–luciferase. Shoot apices isolated as uniformly in size as possible from germinated pea seedlings were frozen after treatment with cryoprotectant to temperatures as low as −50°C and then transferred to liquid nitrogen temperature (−196°C) by a two-step freezing method. The frozen-thawed or unfrozen control shoot apices were washed with fresh medium and an individual shoot apex was suspended in a small volume of distilled water and then heated for 1.5 min on a boiling water bath. To release ATP more completely, the heated shoot apex was rapidly frozen in liquid nitrogen and thawed at room temperature. The content of released ATP was determined with the luciferin-luciferase assay. The percentage of cells in the pea shoot apex which survived freezing and thawing under present conditions was found to be 10% as estimated by this method.